Shogo Atsumi¹, Kazuhisa Miyamoto¹, Kimiko Yamamoto², Hideki Sezutsu³, Isao Kobayashi³, Keiro Uchino³, Toshiki Tamura³, Kazuei Mita², Keiko Kadono-Okuda², Sanae Wada¹, Hiroaki Noda¹

¹Insect-Microbe Research Unit, ²Insect Genome Research Unit, ³Transgenic Silkworm Research Unit

［Abstract］

We identified a gene for recessive form of resistance to Bt toxin, Cry1Ab, on chromosome 15 of the silkworm, Bombyx mori, by positional cloning. The gene, BGIBMGA007792-93, encodes an ATP-binding cassette transporter and is expressed in the midgut. The amino acid sequences of seven resistant silkworm strains revealed a common tyrosine insertion in an outer loop of the predicted transmembrane structure of resistant alleles. We confirmed the role of this ATP-binding cassette transporter gene in Bt resistance by converting a resistant silkworm strain into a susceptible one by using germline transformation. This study represents a direct demonstration of Bt resistance gene function in insects using transgenesis.

［Keywords］

Bt toxin, Cry1Ab, Bombyx mori, resistant gene, ATP-binding cassette transporter

［Background］

The toxins derived from arthropod bacterial pathogen, Bacillus thuringiensis, are widely used as insecticides for insect control or in transgenic crops. The pathogen produces proteinaceous crystalline inclusions, Bt-toxins, which are toxic to some major insect pests such as lepidopteran pests but not to vertebrates (Fig. 1). Increasing use insecticides containing Bt-toxins has threatened to increase the prevalence of Bt resistance among insect pest populations. Using silkworm (Bombyx mori) as a model, we identified a candidate gene for Bt resistance by positional cloning

［Results and Discussion］

1. We chose two strains of silkworm, Chinese no. 2 (C2, resistant) and Ringetsu (Rin, susceptible), which differed by 315 fold in the median lethal concentration (LC50) against Cry1Ab toxin of newly hatched larvae. We first confirmed the linkage assignment of the resistance trait by SNP marker-based analysis. The Bt-resistance trait was located on chromosome 15.
2. We identified a candidate gene, BGIBMGA007792-93, a recessive form of resistance to Cry1Ab toxin on chromosome 15 by positional cloning. The candidate gene was found to encode an ATPbinding cassette transporter and was expressed only in the midgut.
3. We found an insertion of three consecutive nucleotides encoding tyrosine in the candidate gene in resistant strains (Fig. 2). The presence of this common polymorphism in a predicted ABC transporter expressed in larval midgut strongly implicated this gene in contributing to Bt resistance.
4. To confirm that the candidate gene was the causative agent of the Cry1Ab resistance, we introduced a copy of the gene from the susceptible strain into a resistant strain by germline transformation. This resulted in subsequent conversion of the resistant silkworm strain into a susceptible strain thereby confirming a central role for the gene in Cry1Ab toxin action (Fig. 3).

1. Elucidation of the role of ABC transporter as well as the relationship between the insertion of one amino acid (tyrosine) into the amino acid sequences of the ABC transporter and the Cry toxin resistance will contribute in understanding insecticidal action of the Cry toxin.
2. The ABC transporter has already been reported in tobacco budworm (Heliothis virescens), diamondback moth (Plutella xylostella) and cabbage looper (Trichoplusia ni) to be genetically linked with resistance to Cry1Ac. Characterization of Bt toxin resistance genes in insects will facilitate the development of an efficient genetic diagnosis for monitoring the development of Bt resistance in insect pests populations.

Fig.1. Bacillus thuringiensis spores (S) and proteinaceous crystalline inclusions (C)	Fig.2. Sequence alignment of putative amino acids deduced from a portion of gene 007792−93 (from residues 223−246 in C2). Seven Bt resistant strains (upper) and 10 susceptible strains (lower) are shown. Tyrosine is present in resistant strains but lacking in susceptible strains.

Fig.3. Bt toxin bioassay of silkworms transformed with an ABC transporter gene from Bt-susceptible strain for the 4th instar larvae after 1-day-Cry1Ab toxin administration. (A) The larva of resistant strain (52-2 x W1-c) almost completely consumed the toxin treated mulberry leaves. (B) The larva of resistant strain transformed with a susceptible gene derived from Rin (52-2 x SS16-3) died after feeding on toxin treated mulberry leaves.

 

[Reference]

1. Atsumi S, Miyamoto K, Yamamoto K, Narukawa J, Kawai S, Sezutsu H, Kobayashi I, Uchino K, Tamura T, Mita K, Kadono-Okuda K, Wada S, Kanda K, Goldsmith M.R, Noda H (2012) Single amino acid mutation in an ATP-binding cassette transporter gene causes resistance to Bt toxin Cry1Ab in the silkworm, Bombyx mori Proceedings of the National Academy of Sciences USA 109(25):E1591-E1598